| Background and objectiveTherapeutic neovascularization, by administration of exogenous angiogenic growth factors or gene encoding for these, attempts to ameliorate disorders of inadequate tissue perfusion by promoting neovascularization in patients with limb or myocardial ischemia. Therapeutic neovascularization represents an alternative treatment modality for patients with advanced ischemic coronary or peripheral artery occlusive disease. Among the growth factors, hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates genes involved in oxygen homeostasis. Under hypoxia conditions, HIF-1 expression is up-regulated and transactivates hundreds types of its target genes essential for vascular development, erythropoiesis and other pathophysiologic process, induces the development of a functional vascular network. Therefore, HIF-1 has been considered as the most promising angiogenic factor in clinical gene therapy.HIF-1 consists of the O2-regulated HIF-1αsubunit and the constitutively expressed HIF-1βsubunit. HIF-1αis inducible under hypoxia condictions and regulates the activation of HIF-1. However, under normoxic conditions, the residues Pro402 and Pro564 of HIF-1αis hydroxylated by the prolyl hydroxylases, which allows the interaction of HIF-1αwith Von Hippel-Lindau (VHL) and forms the recognization component of an E3 ubiquitin ligase complex, resulting in ubiquitylation and subsequently proteosomal degration. Hydroxylation at residue Asn803 by the action of asparaginyl hydroxylase factor-inhibiting HIF-1 (FIH-1) blocks the binding of HIF-la to its coactivators CBP/p300, leading to inactivation HIF-1α. Therefore, triple point mutant HIF-la bearing mutations Pro402, Pro564 and Asn803 which is not merely stabilization but also constitutive activation was constructed and showed a superior angiogenic potential as compared with either native HIF-la or stable HIF-1αin vitro. However, the in vivo angiogenic effects of triple points mutant HIF-1αhas not been well addressed yet.Ultrasound molecular imaging is a recent development of noninvasive molecular imaging modality and has demonstrated its potential in assessment of tumor or ischemia-mediated angiogenesis on molecular level. Targeted ultrasound imaging involves the design and synthesis of microbubbles that adhere to endothelium under disease-specific conditions, so that the disease can be ultrasonically detected as a prolonged contrast effect that persists in tissue beyond the normal washout time of nontargeted microbubbles. As compared with traditional imaging approaches characterization of diseases by the detection of changes in physics or physiology, targeted contrast imaging could provide capabilities for in vivo ultrasonic detection of phenotypic features of endothelium that predate clinical disease or are otherwise not detectable using currently available technologies.Ultrasound molecular imaging relies on the selective targeting and retention of acoustically active microbubbles at sites of disease. Therefore, it is pivotal to select a proper target and successfully construction of the microbubbles targeted to these molecules. If the microbubbles targeted to specific molecular epitopes expressed in angiogenic endothelium constructed, noninvasive evaluation of angiogenesis could be accomplished. A large number of studies have shown that the molecular expression ofαvβ3-integrin is up-regulated in angiogenic endothelium, which mediates the migration of activated endothelial cells. Therefore,αvβ3-integrin is a particularly attractive candidate for molecular imaging of angiogenesis. The site-targeted microbubbles were prepared by conjugating av-integrin antibody to the surface of a phospholipid microbubble shell via biotin-avidin linkage as described previously and have succeed in imaging matrigel angiogenesis.Ultrasound molecular imaging using site-targeted microbubbles is not only qualitative evaluation of the pathological changes, but also has the potential to quantify the severity of related microvascular endothelium incident. Yeh reported that E-selection expression in lipopolysaccharide induced heart inflammation model was acoustic quantified by using microbubbles targeted to endothelial E-selection. It was also recently reported by us that that the severity of microvascular inflammation could be accurately quantified by using the microbubbles targeted to the endothelial cell adhesion molecule P-selectin with ultrasound imaging.Therefore, in the present study, we will use a novel imaging approaches: ultrasound molecular imaging to evaluate the angiogenic effect of triple mutant HIF-la in a murine model of hind-limb ischemia.Methods1.84 KM female mice (weight 25-30g) under unilateral artery ligation were equally divided into 4 test groups. Adenovirus (Ad)-mediated genes of LacZ (Ad-LZ), Ad-native HIF-1α(Ad-NH), Ad-double mutant HIF-la564/803 (Ad-DM) and Ad-triple mutant HIF-la402/564/803 (Ad-TM) were intramuscularly injected into the ischemia hind-limb of each group, respectively.2. The mRNA expression of HIF-1αas well as its down-streams genes vascular endothelial growth factor (VEGF) and angiopoietin-1 were evaluated by real-time RT-PCR after 3 days of intramuscular gene transfer in skeletal muscles of mice. The protein expression of human HIF-la and mouse VEGF were examined by Western blot after 3 days of intramuscular gene transfer in skeletal muscles of mice.3. Contrast ultrasound perfusion imaging of proximal hind-limb adductor muscles was performed during continuous infusion of general lipid microbubbles in 24 anesthetized mice (n=6 per group) before femoral artery ligation and on postoperative day 0,7,14,21,28.4. The muscle specimens were harvested for evaluation of neovascularization 14 days and 28 days after intramuscular gene transfer, respectively. The densities of capillary and arterioles were analyzed by immunofluorescent using antibodies against BS-1 lectin and a-smooth muscle actin.5. Microbubbles preparation and evaluation in vitro:Targeted microbubbles were prepared by conjugating a biotinylated rat anti-mouse av-intergin antibody to their surface using a biotin-streptavidin linkage (MBαv). A control microbubbles (MBiso), bearing an isotype control antibody, was also constructed. Coulter counter, fluorescence labeling and were used to evaluate the characters and functions of the microbubbles in vitro.6. Ultrasound molecular imaging:on postoperative day 7, ultrasound molecular imaging was performed after intravenous injection of control microbubbles or site-targeted microbubbles in random order with 30min interval.7. Contrast ultrasound imaging analysis:For perfusion imaging, blood flow of both ischemia and contralateral non-ischemia hind-limb was calculated by the product of A andβ; For targeted imaging, skeletal muscles video intensity (VI) was measured by average pixel VI in hind-limb with a low mechanical index (MI) of 0.18 at 8 minutes after injection subtracted from background.8. HE staining and av-integrin immunofluorescent:After targeted imaging,24 experimental mice (n=6 per group) were harvested for HE staining and immunofluorescent analysis of endothelial av-integrin expression.Results1. The mRNA level of HIF-la was found to be 2.9-fold increase in the Ad-TM group as compared with the Ad-LZ group (P=0.000). It was also found to be 2.4-fold relative to the Ad-NH group (P=0.008) and 1.7-fold relative to the Ad-DM group (P =0.055), respectively. The mRNA expression of angiopoietin-1 induced by Ad-TM was found to be significantly increase by 1.4-2.2 (P<0.05) fold as compared to other control groups, respectively. The mRNA expression of VEGF was significantly increase in the Ad-TM group as compared with Ad-LZ group (P=0.007), however, there was no statistical significance of VEGF mRNA expression between Ad-NH group, Ad-DM group and Ad-TM group.2. Human HIF-la protein could not be found in mice treated with Ad-LacZ but was present in mice treated with three isoforms of Ad-HIF-la. It was significantly increased in the Ad-TM group as compared with other control groups (F= 102.943, P=0.000). The total VEGF protein level from the ischemia adductor muscles also followed a similar pattern as VEGF mRNA level. Significantly greater VEGF protein was found in the Ad-TM group as compared with other control groups (F=38.104, P=0.000).3. Immediately after femoral artery ligation, the blood flow ratio of the ischemic/ non-ischemia hind-limb was reduced to (16.9±2.7)% in Ad-LZ group, (15.2±3.0)% in Ad-NH group, (15.9±2.1)% in Ad-DM group and (16.0±2.4)% in Ad-TM group (F=38.104, P=0.000). Although the blood flow gradually recovered after femoral artery ligation, the blood flow ratio was persistently higher in mice receiving Ad-TM as compared with any of the control group during the follow-up period. On postoperative day 28, the blood flow ratio was significantly increase in the Ad-TM group (89.3±5.4)% as compared with Ad-LZ group(57.0±4.6)%,Ad-NH group (62.3±3.8)% and Ad-DM group (73.4±6.0)%, (F=53.770, P=0.000)。4. BS-1 lectin staining was performed to label capillary. Capillaries density (expressed as capillary/muscle fiber) on postoperative day 14 for the Ad-TM group was 1.42±0.21, much superior (F=44.829, P=0.000) to that of the Ad-DM group (1.05±0.08), the Ad-NH group (0.77±0.08) and the Ad-LZ group (0.56±0.06). The outcome sustained on postoperative day 28, the capillaries in the Ad-TM group (2.06±0.23) exceeded (F=74.693, P=0.000) that of the Ad-DM group (1.47±0.17), Ad-NH group (1.01±0.14) and Ad-LZ group (0.8±0.08).α-smooth muscle actin staining was also performed to label arterioles. On postoperative day 14, the arterioles density (expressed as the number of arteries per high power fields) of the Ad-TM group (7±1.58) was significantly higher (F=7.965, P=0.002) than any other control group (2.4±1.14 for the Ad-LZ group,3.6±1.14 for the Ad-NH group and 4±2.12 for the Ad-DM group). There was a progressive increase in the number of arteries in the ischemia adductor muscles observed in all groups, however, it is was also dramatically higher (F=21.284, P=0.000) in the Ad-TM group (15.2±2.59) as compared with the Ad-DM group (10.4±1.95), the Ad-NH group (7.4±1.14) and the Ad-LZ group (6.4±1.67). Taken together, these data demonstrated that HIF-1αrestored blood flow through angiogenesis and arteriogenesis and Ad-TM is essential for optimal HIF-mediated angiogenic effects. 5. The concentration of microbubbles for MBav and MBiso measured by Coulter Counter were 1.4±0.39×108/ml and 1.5±0.31×108个/ml respectively. The size distribution for MBav and MBiso measured by Coulter Counter were 2.08±0.85μm and 2.07±1.13μm. Both the rat anti-mouse av-intergin antibody and the isotype control antibody linked well to the surface of microbubbles, which were observed with fluorescence microscopy.6. With ultrasound molecular imaging, there was minimal signal for MBiso in both ischemia and non-ischemia hind-limbs. In comparison, there was strong signal enhancement from MBav in the ischemia hind-limb, with only minimal signal in the non-ischemia hind-limb. Signal enhancement for av-targeted microbubbles increased incrementally from Ad-LZ treated mice to Ad-NH treated mice to Ad-DM treated mice to Ad-TM treated mice.7. The quantitative data showed that there was no significant difference of VI with MBav (3.75±1.19)U and MBiso (3.80±1.08)U in non-ischemia muscles, (t=-0.300, P=0.776).VI of the ischemic muscle with MBav in all test groups was significantly higher than that with MBiso [(10.9±2.0)U vs.(4.9±1.1)U for Ad-LZ group, t=6.946, P=0.001; (14.1±2.8)U vs.(4.9±1.6)U for Ad-NH group t=13.010, P=0.009; (18.8±3.5)U vs.(6.2±1.4)U for Ad-DM group, t=11.408, P=0.000; (25.7±3.1)U vs.(5.2±1.8)U for Ad-TM group, t=13.876, P=0.000]. The VI for MBav in Ad-TM group was obviously higher (F=58.110, P=0.000) than that of Ad-DM group, Ad-NH group, Ad-LZ group and non-ischemia group while there was no significantly difference (F=2.223, P=0.095) of VI for MBiso in all groups.8. HE staining showed that skeletal muscle cells in non-ischemia hind-limb were normal, while in ischemia hind-limb, they were edema and infiltrated with a large number of leukocytes. The av-intergin in the non-ischemia muscles was below the level of detection, whereas in the ischemia muscles, av-intergin was observed in some capillary sprouts. The capillaries expressing av-intergin was enhanced in the Ad-TM group as compared with any other control group. Conclusions1. Ad-HIF-1αpromotes blood flow through angiogenesis and arteriogenesis, and Ad-TM has an optimal HIF-mediated angiogenic effects in murine model of hind-limb ischemia.2. Microbubbles targeted to integrins av-integrin can be successfully constructed by combinding anti av-integrin monoclone antibodies to lipid microbubbles via "avidin-biotin" bridging chemistry.3. Ultrasound molecular imaging with microbubbles targeted to av-integrin can quantify the HIF-1αmediated neovascularization.
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